Mri and optical assays for proteases

Abstract

The present invention provides multifunctional nanoplatforms for assessing the activity of a protease in vivo or in vitro, along with methods of imaging and detecting the presence of cancerous or precancerous tissues, and the therapeutic treatment thereof, including monitoring of treatment. The diagnostic nanoplatforms comprise nanoparticles and are linked to each other or other particles via an oligopeptide linkage that comprises a consensus sequence specific for the target protease. Cleavage of the sequence by the target protease can be detected using various sensors, and the diagnostic results can be correlated with cancer prognosis. Individual unlinked nanoplatforms are also adaptable for therapeutic hyperthermia treatment of the cancerous tissue.

1 . A nanoplatform assembly for detecting protease activity comprising:
 a first nanoplatform comprising a first nanoparticle and a protective layer;   a second nanoplatform comprising a second nanoparticle and a protective layer; and   an oligopeptide linkage between said first and second nanoplatforms, said linkage comprising a protease consensus sequence,   wherein at least one of said first or second nanoplatforms further comprises a functional group selected from the group consisting of porphyrins, chlorins, bacteriochlorins, phthalocyanines, biotin, derivatives thereof, and combinations thereof.   
     
     
         2 . The nanoplatform assembly of  claim 1 , wherein said first nanoparticle and second nanoparticle are respective core/shell nanoparticles. 
     
     
         3 . The nanoplatform assembly of  claim 2 , wherein each core is individually selected from the group consisting of Au, Ag, Cu, Co, Fe, and Pt. 
     
     
         4 . The nanoplatform assembly of  claim 3 , wherein said core is a strongly paramagnetic Fe core. 
     
     
         5 . The nanoplatform assembly of  claim 2 , wherein each shell is individually selected from the group consisting of Au, Ag, Cu, Co, Fe, Pt, the metal oxides thereof, and combinations thereof. 
     
     
         6 . The nanoplatform assembly of  2 , wherein said shell comprises iron oxide. 
     
     
         7 . The nanoplatform assembly of  claim 1 , wherein said first and second nanoparticles have a Brunauer-Emmett-Teller multipoint surface area of at least about 20 m 2 /g. 
     
     
         8 . The nanoplatform assembly of  claim 1 , said protective layers being individually selected from the group consisting of siloxane nanolayers, ligand monolayers, and combinations thereof. 
     
     
         9 - 19 . (canceled) 
     
     
         20 . The nanoplatform assembly of  claim 1 , wherein said protease consensus sequence is selected from the group consisting of SGRSA (SEQ ID NO: 2), VPMSMRGG (SEQ ID NO: 3), IPVSLRSG (SEQ ID NO: 4), RPFSMIMG (SEQ ID NO: 5), VPLSLTMG (SEQ ID NO: 6), VPLSLYSG (SEQ ID NO: 7), IPESLRAG (SEQ ID NO: 8), SGSPAFLAKNR (SEQ ID NO: 9), DAFK (SEQ ID NO: 10), SGKPILFFRL (SEQ ID NO: 11), SGKPIIFFRL (SEQ ID NO:12), GPLGMLSQ (SEQ ID NO:13), HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO: 25), GPQGLAGQRGIV (SEQ ID NO: 26), SLLKSRMVPNFN (SEQ ID NO: 27), SLLIFRSWANFN (SEQ ID NO: 28), SGVVIATVIVIT (SEQ ID NO: 29), GAANLVRG (SEQ ID NO: 74), and PRAGA(SEQ ID NO: 75). 
     
     
         21 .- 22 . (canceled) 
     
     
         23 . A composition comprising a diagnostic assay including the assembly of  claim 1  and a pharmaceutically-acceptable carrier. 
     
     
         24 .- 54 . (canceled) 
     
     
         55 . An MRI imaging method for detecting the activity of a protease associated with a cancerous or precancerous cell in a mammal comprising:
 (a) administering to the mammal the composition of  claim 23 ;   (b) locating said assay in a region of interest in the mammal suspected of having a cancerous or precancerous cell;   (c) transmitting radio frequency pulses to said region of interest; and   (d) acquiring MR image data of the region of interest, said MR image data comprising T 1  and T 2  values.   
     
     
         56 .- 60 . (canceled) 
     
     
         61 . The method of  claim 55 , said MR image data being T 2 -weighted, said method further comprising detecting a change in the acquired T 2  values over time, said change corresponding to protease activity. 
     
     
         62 .- 66 . (canceled) 
     
     
         67 . An MRI imaging method for detecting the activity of a protease associated with a cancerous or precancerous cell in a mammal comprising:
 (a) administering to the mammal a composition comprising a diagnostic assay including the nanoplatform assembly of  claim 1 , wherein said protease consensus sequence is SGRSA (SEQ ID NO: 2).   (b) locating said assay in a region of interest in the mammal suspected of having a cancerous or precancerous cell;   (c) transmitting radio frequency pulses to said region of interest; and   (d) acquiring a first MR image data of the region of interest, said first MR image data comprising T 1  and T 2  values.   
     
     
         68 . The method of  claim 67 , wherein said first MR image data indicates protease activity, said method further comprising:
 (e) administering to the mammal a composition comprising a diagnostic assay including the nanoplatform assembly of  claim 1 , wherein said protease consensus sequence is VPLSLTMG (SEQ ID NO: 6).   (f) locating said assay in a region of interest in the mammal suspected of having a cancerous or precancerous cell;   (g) transmitting radio frequency pulses to said region of interest; and   (h) acquiring a second MR image data of the region of interest, said second MR image data comprising T 1  and T 2  values.   
     
     
         69 . The method of  claim 68 , wherein said second MR image indicates protease activity, said activity being correlated to a prognosis for angiogenesis or metastasis. 
     
     
         70 . The method of  claim 69 , further comprising:
 (i) administering to the mammal a composition comprising a diagnostic assay including the nanoplatform assembly of  claim 1 , wherein said protease consensus sequence is VPMSMRGG (SEQ ID NO: 3),   (j) locating said assay in a region of interest in the mammal suspected of having a cancerous or precancerous cell;   (k) transmitting radio frequency pulses to said region of interest; and   (l) acquiring a third MR image data of the region of interest, said third MR image data comprising T 1  and T 2  values.   
     
     
         71 .- 75 . (canceled) 
     
     
         76 . A nanoplatform comprising a first nanoparticle and a protective layer surrounding said nanoparticle, said protective layer being selected from the group consisting of siloxane nanolayers, ligand monolayers, gold coating layer, and combinations thereof. 
     
     
         77 . The nanoplatform of  claim 76 , further comprising a functional group selected from the group consisting of porphyrins, chlorins, bacteriochlorins, phthalocyanines, biotin, derivatives thereof, and combinations thereof. 
     
     
         78 . The nanoplatform of  claim 76 , said protective layer comprising a siloxane nanolayer, wherein said nanoplatform further comprises a ligand monolayer surrounding said siloxane nanolayer. 
     
     
         79 . The nanoplatform of  claim 78 , further comprising a gold coating layer surrounding said ligand monolayer. 
     
     
         80 . The nanoplatform of  claim 76 , wherein said nanoparticle is a core/shell nanoparticle, said core being selected from the group consisting of Au, Ag, Cu, Co, Fe, and Pt, and said shell being selected from the group consisting of Au, Ag, Cu, Co, Fe, Pt, the metal oxides thereof, and combinations thereof. 
     
     
         81 . The nanoplatform of  claim 80 , wherein said core is a strongly paramagnetic Fe core. 
     
     
         82 .- 83 . (canceled) 
     
     
         84 . The nanoplatform of  claim 76 , wherein said nanoplatform is linked via an oligopeptide linkage to a particle selected from the group consisting of chromophores/luminophores, quantum dots, viologens, and combinations thereof, said oligopeptide linkage comprising a protease consensus sequence. 
     
     
         85 .- 91 . (canceled) 
     
     
         92 . The nanoplatform of  claim 76 , said nanoplatform being unlinked to any other nanoplatform. 
     
     
         93 . (canceled) 
     
     
         94 . A composition comprising a diagnostic assay including the nanoplatform of  claim 76  and a pharmaceutically-acceptable carrier. 
     
     
         95 .- 97 . (canceled) 
     
     
         98 . A method of inhibiting the growth of cancerous or precancerous cells in a mammal comprising:
 (a) administering to the mammal the composition of  claim 94 ;   (b) locating said assay in a region of interest in the mammal suspected of having a cancerous or precancerous cell; and   (c) heating said nanoplatform using magnetic A/C-excitation, whereby the tissue in said region of interest is heated to a temperature of at least about 40° C., wherein said heating (c) results in apoptosis of said cancerous or precancerous cells.   
     
     
         99 .- 113 . (canceled) 
     
     
         114 . An MRI contrast agent comprising a core/shell nanoparticle having an iron core, said MRI contrast agent having an r 1  of greater than about 100 mM −1 s −1  for T 2 -enhancement and an r 2  with an integer number greater than about −2,000 mM −1 s −1  for T 2 -decrease.